In recent years, the diabetic population has increased steadily and diabetes is attracting attention as one of adult diseases. Non-insulin-dependent diabetes mellitus (NIDDM) is a type of diabetes frequently found in Japan and its early detection and timely treatment are necessary to prevent the disease. Although the factors causative of NIDDM have not been fully elucidated, recent advances have provided important new insights into this process.
It is now well established that abnormalities in insulin sensitive mechanisms and reduced secretion of insulin are causes of insufficient insulin activity in NIDDM. In Europe and America, insulin resistance is predominant in patients with NIDDM while, in Japan, insulin hyposecretion is often the major cause. With recent advances in molecular biology, the cellular and molecular mechanisms underlying insulin resistance such as the insulin receptor structure and the mechanism of signal transduction downstream of the receptor, have been investigated in detail. During the last decade, glucose transporter genes has been cloned and the relationship between mutations in the genes and the process of diabetes has been studied. However, the insulin, glucokinase and mitochondrial gene abnormalities so far elucidated, taken together, account for not more than 1% of NIDDM cases. Other gene abnormalities are to be revealed in the future.
In recent years, antidiabetic agents quite differing from the conventional oral hypoglycemic agents in the mechanism of action, such as the .alpha.-glycosidase inhibitors acarbose and voglibose (Diabetes Frontier, 3, 557-564 (1992); Drugs, 46, 1025-1054 (1994); Igaku no Ayumi, 149, 591-618 (1989); Rinsho to Kenkyu (Japan. J. Clinics Exper. Med.), 67, 219-233 (1990); Rinsho to Kenkyu, 69, 919-932 (1992); Rinshoi (Clinical Medicine), 21 (supplement), 578-587 (1995)) and the insulin resistance improving agents, troglitazone and pioglitazone, (Diabetes, 37, 1549-1558 (1998); Rinsho Iyaku, 9 (supplement 3), 127-150 (1993); New Engl. J. Med., 331, 1188-1193 (1994); "Atarashii Tonyobyo Chiryoyaku (New Antidiabetics)" (edited by Yoshio Goto), published by Iyaku Journal Co., Osaka, (1994)) have been developed. They are expected to be soon on the market.
Meanwhile, in the United States, a biguanide derivative was approved in 1996 as an antidiabetic for general prescription (New Engl. J. Med., 333, 541-549 (1995); Diabetes Spectrum, 8, 194-197 (1995)) and is attracting attention. The above-mentioned drugs, unlike sulfonylureas (SUs) which have been used for many years in routine medical care, produce a hypoglycemic effect without promoting insulin secretion from .beta. cells of the pancreas.
It is considered, at present, that there are nine mechanisms through which antidiabetics might be able to improve insulin resistance as follows: (1) activation of insulin receptor kinase, (2) promotion of translocation of glucose transporters, (3) correction of the action of the rate-limiting enzyme involved in glucose metabolism and correction of abnormalities in glucose metabolism, (4) inhibition of gluconeogenesis in liver, (5) promotion of glucose uptake by liver, (6) enhancement of glycogenesis in liver, (7) reduction in blood lipid level, (8) decrease in gluconeogenesis in liver as resulting from the reduction in blood lipid level, and (9) enhancement of insulin sensitivity as resulting from the reduction in blood lipid level.
GFAT is an important enzyme catalyzing the conversion of fructose-6-phosphate to glucosamine-6-phosphate, which is the rate-limiting step in the hexosamine biosynthesis pathway. Inhibitors of GFAT activity are thought to promote glucose influx by cells and thereby reducing the blood glucose level. Therefore, these inhibitors are expected to be of use as antidiabetics. Their mechanism of action is thought to be associated with the process (2) or (5) mentioned above.
While the hexosamine biosynthesis pathway metabolizes glucosamine-6-phosphate to UDP-N-acetylglucosamine, CMP-N-acetylneuraminic acid, etc., those metabolic intermediates are thought to be utilized as precursors for glycosylation of proteins or as essential substrates for the synthesis of proteoglycan and ganglioside.
Insulin activates its signal transduction pathway through binding insulin receptor and translocates glucose transporters (GLUT4 etc.) pooled within cells to the cell membrane resulting in increasing glucose influx. Glucose is metabolized by glycolysis pathway and ATP is accumulated as an energy source. When the influx of glucose is excessive, however, fructose-6-phosphate enters the hexosamine biosynthesis pathway and is converted to glucosamine-6-phosphate catalyzed by GFAT. Although detailed mechanisms remain unknown, several observations indicate that metabolites of glucosamine-6-phosphate prevent glucose transporters from translocating to cell membrane, resulting in reducing cellular glucose influx (FASEB J., 5, 3031-3036 (1991); Diabetologia, 38, 518-524 (1995); J. Biol. Chem., 266, 10115-10161 (1991): J. Biol. Chem., 266, 4706-4712 (1991); Endocrinology, 136, 2809-2816 (1995)).
Therefore, the hexosamine biosynthesis pathway is considered to control the influx of glucose by a feed-back manner. GFAT is the rate-limiting enzyme in this pathway. GFAT activity is also known to be generally high in patients with NIDDM and is considered to be one of the causes of high blood glucose levels (Diabetes, 45, 302-307 (1996)).
Hypoglycemic agents, such as inhibitors of GFAT activity, whose action is mainly directed to some other tissues than pancreas invariably improve insulin resistance in target tissues. These agents have some clinical merits in addition to their hypoglycemic activity, because of their secondary effects. When used in combination with other drugs, they are highly effective and have very bright prospects before them.
Recently a human GFAT gene has been cloned (J. Biol. Chem., 267, 25208-25212 (1992)). The gene product is a 77 kDa protein composed of 681 amino acid residues. GFAT genes have been cloned from other animal species as well. For example, a murine GFAT is highly homologous to the human GFAT (91% at the nucleotide level and 98.6% at the amino acid level), hence it is considered to be the counterpart of the human GFAT (Gene, 140, 289-290 (1994)). In addition, a yeast GFAT (J. Biol. Chem., 264, 8753-8758 (1989)) and a Escherichia coli-derived GFAT (Biochem. J., 224, 779-815 (1984)) have also been reported, each having high homology with the human GFAT. It is not known about the occurrence of a new GFAT isoform gene.
Isolation of a novel protein showing GFAT activity, if successful, would make it possible to perform further investigations to elucidate the regulatory function of GFAT in the hexosamine biosynthesis pathway and, if its expression is tissue-specific, to elucidate the tissue-specific mechanisms of glucose metabolism. A specific inhibitor of the novel GFAT protein, if developed, would make it possible to develop a hypoglycemic agent acting through novel mechanisms of action and contributing to the prevention and treatment of diabetes and diabetic complications without producing serious adverse effects.
As the result of intensive research, the inventors of the present invention succeeded in cloning a cDNA having a novel nucleotide sequence from a human brain-derived cDNA library and found that the protein encoded thereby has GFAT activity. As the result of continued investigations based on such findings, the present inventors have now completed the present invention.